Elevated blood plasma homocysteine levels can be correlated to risk of cardiovascular disease, e.g. coronary heart disease, coronary artery disease, cerebrovascular disease, and peripheral vascular disorders. Indeed elevated homocysteine levels are thought to be a better predictor of cardiovascular disease than elevated cholesterol levels. Generally plasma levels of 15 μM and below are considered healthy (see for example New England Journal of Medicine 1997, 337: 230).
Accordingly there is a need for reliable methods for determining homocysteine levels in patients.
Direct determination of homocysteine has thus far proved to be complicated, e.g. because it has not been found possible to raise an antibody against homocysteine (HCy) which is not cross-reactive with other substances present in biological samples.
However, in WO 93/15520 (Axis) and WO 98/07872 (Glasgow) for example, homocysteine assays are described which involve enzymatic conversion of homocysteine and determination of homocysteine levels by determination of a homocysteine conversion product produced by the enzyme-mediated conversion.
Such assays require the use of a reducing agent (e.g. dithiothreitol) to liberate covalently bound homocysteine and of a homocysteine converting enzyme and while they perform well there is room for improvement in terms of increasing signal to noise, e.g. by reducing background signal.